Romaine lettuce cultivar toro

ABSTRACT

The invention provides seed and plants of the romaine lettuce cultivar designated cv. Toro. The invention thus relates to the plants, seeds and tissue cultures of romaine lettuce cv. Toro, and to methods for producing a lettuce plant produced by crossing a plant of romaine lettuce cv. Toro with itself or with another lettuce plant, such as a plant of another line. The invention further relates to seeds and plants produced by such crossing. The invention further relates to parts of a plant of romaine lettuce line cv. Toro, including the gametes of such plants.

FIELD

The present invention relates to the field of plant breeding and, morespecifically, to the development of romaine lettuce cultivar (cv.) Toro.

BACKGROUND

The goal of vegetable breeding is to combine various desirable traits ina single variety/hybrid. Such desirable traits may include greateryield, resistance to insects or pests, tolerance to heat and drought,better agronomic quality, higher nutritional value, growth rate andfruit properties.

Breeding techniques take advantage of a plant's method of pollination.There are two general methods of pollination: a plant self-pollinates ifpollen from one flower is transferred to the same or another flower ofthe same plant or plant variety. A plant cross-pollinates if pollencomes to it from a flower of a different plant variety.

Plants that have been self-pollinated and selected for type over manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny, a homozygous plant. A crossbetween two such homozygous plants of different varieties produces auniform population of hybrid plants that are heterozygous for many geneloci. Conversely, a cross of two plants each heterozygous at a number ofloci produces a population of hybrid plants that differ genetically andare not uniform. The resulting non-uniformity makes performanceunpredictable.

The development of uniform varieties requires the development ofhomozygous inbred plants, the crossing of these inbred plants, and theevaluation of the crosses. Pedigree breeding and recurrent selection areexamples of breeding methods that have been used to develop inbredplants from breeding populations. Those breeding methods combine thegenetic backgrounds from two or more plants or various other broad-basedsources into breeding pools from which new lines are developed byselfing and selection of desired phenotypes. The new lines are evaluatedto determine which of those have commercial potential.

SUMMARY

In one aspect, the present invention provides a romaine lettuce plantdesignated cv. Toro. Also provided are lettuce plants having thephysiological and morphological characteristics of the romaine lettucedesignated cv. Toro. Parts of the romaine lettuce plant of the presentinvention are also provided, for example, including pollen, an ovule,and a cell of the plant.

The invention also concerns seed of romaine lettuce cv. Toro. Thelettuce seed of the invention may be provided as an essentiallyhomogeneous population of romaine lettuce seed of cv. Toro. Essentiallyhomogeneous populations of seed are generally free from substantialnumbers of other seed. In certain embodiments of the invention, seed ofcv. Toro may be provided forming at least about 97% of the total seed,including at least about 98%, 99%, or more of the seed. The populationof lettuce seed may be particularly defined as being essentially freefrom hybrid seed. The seed population may be separately grown to providean essentially homogeneous population of romaine lettuce plantsdesignated cv. Toro.

In another aspect of the invention, a plant of romaine lettuce cv. Torocomprising an added heritable trait is provided. The heritable trait maycomprise a genetic locus that is a dominant or recessive allele. In oneembodiment of the invention, a plant of romaine lettuce cv. Toro isdefined as comprising a single locus conversion. In specific embodimentsof the invention, an added genetic locus confers one or more traits suchas, for example, herbicide tolerance, insect resistance, diseaseresistance, and modified carbohydrate metabolism. The trait may be, forexample, conferred by a naturally occurring gene introduced into thegenome of the line by backcrossing, a natural or induced mutation, or atransgene introduced through genetic transformation techniques into theplant or a progenitor of any previous generation thereof. Whenintroduced through transformation, a genetic locus may comprise one ormore transgenes integrated at a single chromosomal location.

In another aspect of the invention, a tissue culture of regenerablecells of a plant cv. Toro is provided. The tissue culture willpreferably be capable of regenerating plants capable of expressing allof the physiological and morphological characteristics of the line, andof regenerating plants having substantially the same genotype as otherplants of the line. Examples of some of the physiological andmorphological characteristics of the cv. Toro include those traits setforth in the tables herein. The regenerable cells in such tissuecultures may be derived, for example, from embryos, meristems,cotyledons, pollen, leaves, anthers, roots, root tips, pistil, flower,seed and stalks. Still further, the present invention provides lettuceplants regenerated from a tissue culture of the invention, the plantshaving all the physiological and morphological characteristics of cv.Toro.

In yet another aspect of the invention, processes are provided forproducing lettuce seeds and plants, which processes generally comprisecrossing a first parent lettuce plant with a second parent lettuceplant, wherein at least one of the first or second parent lettuce plantsis a plant of cv. Toro. These processes may be further exemplified asprocesses for preparing hybrid lettuce seed or plants, wherein a firstlettuce plant is crossed with a second lettuce plant of a different,distinct line to provide a hybrid that has, as one of its parents, theromaine lettuce plant cv. Toro. In these processes, crossing will resultin the production of seed. The seed production occurs regardless ofwhether the seed is collected or not.

In one embodiment of the invention, the first step in “crossing”comprises planting seeds of a first and second parent lettuce plant,often in proximity so that pollination will occur for example, mediatedby insect vectors. Alternatively, pollen can be transferred manually.Where the plant is self-pollinated, pollination may occur without theneed for direct human intervention other than plant cultivation.

A second step may comprise cultivating or growing the seeds of first andsecond parent lettuce plants into plants that bear flowers. A third stepmay comprise preventing self-pollination of the plants, such as byemasculating the male portions of flowers, (i.e., treating ormanipulating the flowers to produce an emasculated parent lettuceplant). Self-incompatibility systems may also be used in some hybridcrops for the same purpose. Self-incompatible plants still shed viablepollen and can pollinate plants of other varieties but are incapable ofpollinating themselves or other plants of the same line.

A fourth step for a hybrid cross may comprise cross-pollination betweenthe first and second parent lettuce plants. Yet another step comprisesharvesting the seeds from at least one of the parent lettuce plants. Theharvested seed can be grown to produce a lettuce plant or hybrid lettuceplant.

The present invention also provides the lettuce seeds and plantsproduced by a process that comprises crossing a first parent lettuceplant with a second parent lettuce plant, wherein at least one of thefirst or second parent lettuce plants is a plant designated cv. Toro. Inone embodiment of the invention, lettuce seed and plants produced by theprocess are first generation (F₁) hybrid lettuce seed and plantsproduced by crossing a plant in accordance with the invention withanother, distinct plant. The present invention further contemplatesplant parts of such an F₁ hybrid lettuce plant, and methods of usethereof. Therefore, certain exemplary embodiments of the inventionprovide an F₁ hybrid lettuce plant and seed thereof.

In still yet another aspect of the invention, the genetic complement ofthe romaine lettuce plant designated cv. Toro is provided. The phrase“genetic complement” is used to refer to the aggregate of nucleotidesequences, the expression of which sequences defines the phenotype of,in the present case, a lettuce plant, or a cell or tissue of that plant.A genetic complement thus represents the genetic makeup of a cell,tissue or plant, and a hybrid genetic complement represents the geneticmake-up of a hybrid cell, tissue or plant. The invention thus provideslettuce plant cells that have a genetic complement in accordance withthe lettuce plant cells disclosed herein, and plants, seeds and plantscontaining such cells.

Plant genetic complements may be assessed by genetic marker profiles,and by the expression of phenotypic traits that are characteristic ofthe expression of the genetic complement, e.g., isozyme typing profiles.It is understood that cv. Toro or a first generation progeny thereofcould be identified by any of the many well-known techniques such as,for example, Simple Sequence Length Polymorphisms (SSLPs) (Williams etal., Nucleic Acids Res., 1 8:6531-6535, 1990), Randomly AmplifiedPolymorphic DNAs (RAPDs), DNA Amplification Fingerprinting (DAF),Sequence Characterized Amplified Regions (SCARs), Arbitrary PrimedPolymerase Chain Reaction (AP-PCR), Amplified Fragment LengthPolymorphisms (AFLPs) (EP 534 858, specifically incorporated herein byreference in its entirety), and Single Nucleotide Polymorphisms (SNPs)(Wang et al., Science, 280:1077-1082, 1998).

In still yet another aspect, the present invention provides hybridgenetic complements, as represented by lettuce plant cells, tissues,plants, and seeds, formed by the combination of a haploid geneticcomplement of a lettuce plant of the invention with a haploid geneticcomplement of a second lettuce plant, preferably, another, distinctlettuce plant. In another aspect, the present invention provides alettuce plant regenerated from a tissue culture that comprises a hybridgenetic complement of this invention.

In still yet another aspect, the invention provides a method ofdetermining the genotype of a plant of romaine lettuce cv. Torocomprising detecting in the genome of the plant at least a firstpolymorphism. The method may, in certain embodiments, comprise detectinga plurality of polymorphisms in the genome of the plant. The method mayfurther comprise storing the results of the step of detecting theplurality of polymorphisms on a computer readable medium. The inventionfurther provides a computer readable medium produced by such a method.

In still yet another aspect, the present invention provides a method ofproducing a plant derived from cv. Toro, the method comprising the stepsof: (a) preparing a progeny plant derived from cv. Toro, wherein saidpreparing comprises crossing a plant of the cv. Toro with a secondplant; and (b) crossing the progeny plant with itself or a second plantto produce a seed of a progeny plant of a subsequent generation. Infurther embodiments, the method may additionally comprise: (c) growing aprogeny plant of a subsequent generation from said seed of a progenyplant of a subsequent generation and crossing the progeny plant of asubsequent generation with itself or a second plant; and repeating thesteps for an additional 3-10 generations to produce a plant derived fromcv. Toro. The plant derived from cv. Toro may be an inbred line, and theaforementioned repeated crossing steps may be defined as comprisingsufficient inbreeding to produce the inbred line. In the method, it maybe desirable to select particular plants resulting from step (c) forcontinued crossing according to steps (b) and (c). By selecting plantshaving one or more desirable traits, a plant derived from cv. Toro isobtained which possesses some of the desirable traits of the line aswell as potentially other selected traits.

In certain embodiments, the present invention provides a method ofproducing lettuce comprising: (a) obtaining a plant of romaine lettucecv. Toro, wherein the plant has been cultivated to maturity, and (b)collecting lettuce from the plant.

Any embodiment discussed herein with respect to one aspect of theinvention applies to other aspects of the invention as well, unlessspecifically noted.

The term “about” is used to indicate that a value includes the standarddeviation of error for the device or method being employed to determinethe value. The use of the term “or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only orthe alternatives are mutually exclusive, although the disclosuresupports a definition that refers to only alternatives and to “and/or.”When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more,”unless specifically noted. The terms “comprise,” “have” and “include”are open-ended linking verbs. Any forms or tenses of one or more ofthese verbs, such as “comprises,” “comprising,” “has,” “having,”“includes” and “including,” are also open-ended. For example, any methodthat “comprises,” “has” or “includes” one or more steps is not limitedto possessing only those one or more steps and also covers otherunlisted steps. Similarly, any plant that “comprises,” “has” or“includes” one or more traits is not limited to possessing only thoseone or more traits and covers other unlisted traits.

Other objects, features, and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and any specificexamples provided, while indicating specific embodiments of theinvention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1. Mature leaf of cultivar Toro.

FIG. 2. Fourth leaf of cultivar Toro.

FIG. 3. Pedigree of cultivar Toro.

DETAILED DESCRIPTION

The invention provides methods and compositions relating to plants,seeds and derivatives of romaine lettuce cv. Toro. This line showsuniformity and stability within the limits of environmental influencefor the traits described hereinafter. Lettuce cv. Toro providessufficient seed yield. By crossing with a distinct second plant, uniformF1 hybrid progeny can be obtained.

In another embodiment, the invention provides a vigorous romaine lettucecultivar adapted for production in the Central California (CA) coastalregion, as well as Imperial County, Calif. and Yuma County, Ariz. Thesowing dates for this variety are all 12 months. Cv. Toro was selectedfor medium to large size with good weight, having resistance to lettucenecrotic stunt virus (aka, tomato bushy stunt virus) and tipburn,compared to the most similar commercial cultivar, cv. River Road, aswell as cvs. Lompoc and Ranger.

The areas of adaptation of cv. Toro include Santa Cruz, San Benito,Monterey, San Luis Obispo, Santa Barbara, Ventura, Riverside, ImperialCounties of California, and Yuma County of Arizona.

A. Origin and Breeding History of Lettuce Cv. Toro

The creation of cv. Toro began in 2014 with the cross between cv.Klamath and cv. Sharp Shooter. The female was cv. Klamath (romaine) andthe male cv. Sharp Shooter (iceberg), both sold in the past by SeminisVegetable Seeds. Single plant selections were made in subsequent yearsuntil the F6 generation in 2017, when two lines were selected out ofline 801116→801116-1 and 801116-5, for potential commercial release. Thenext year, 2017, line 801116-1 was re-selected to create two new lines801116-1→801116-1-3 and 801116-1-5 for commercial release andsubsequently, three lines were developed as separate lines. In 2020, thethree lines were named as cv. Toro (801116-5-1), cv. Lompoc(801116-1-3), and cv. Ranger (801116-1-5). Field trials of cv. Toro werecarried out in both 2019 and 2020, using Fg and F9 seed.

The breeding work was conducted by Dr. William Waycott of Nipomo NativeSeeds, LLC, Nipomo, Calif. Replicated field trials were placed incommercial production areas of California during 2019 and 2020.

B. Physiological and Morphological Characteristics of Romaine LettuceCv. Toro

In accordance with one aspect of the present invention, there isprovided a plant having the physiological and morphologicalcharacteristics of lettuce cv. Toro. A description of the physiologicaland morphological characteristics of lettuce cv. Toro is presented inTable 1.

TABLE 1 Physiological and Morphological Characteristics of RomaineLettuce cv. Toro and cv. River Road Romaine Lettuce cv. Toro cv. RiverCharacteristic (No. T0047-904301) Road 1-x plant type Green Romaine SEED2-1 seed color black 2-x light dormancy light not required 2-x heatdormancy susceptible SEEDLING x-2 seedling anthocyanin absent x-3cotyledon size medium x-4 cotyledon shape medium eliptical 3-x cotyledonshape intermediate FOURTH LEAF 3-x fourth leaf shape elongated 3-xfourth leaf 3-x fourth leaf, length/width ratio 3.0 3-x fourth leaf,apical margin entire 3-x fourth leaf, basal margin entire 3-x fourthleaf, undulation slight 3-x fourth leaf, green color green 3-x fourthleaf, anthocyanin distribution N/A 3-x fourth leaf, anthocyaninconcentration N/A 3-x fourth leaf, rolling absent 3-x fourth leaf,cupping uncupped 3-x fourth leaf, reflexing none 10-12 LEAF x-5 matureleaf, attitude erect x-6 mature leaf, blade divisions entire MATUREPLANT 4-x mature leaf x-28 mature leaf, incisions, apical part presentabsent 4-29 mature leaf, incisions depth, apical part shallow absentx-30 mature leaf, incisions density, apical part sparse absent x-31mature leaf, incisions type, apical part sinuate absent x-32 matureleaf, venation (flabellate = ginkgo) not flabellate not flabellate 4-xmature leaf, indentation crenate crenate 4-27 mature leaf, undulation,apical part absent weak 4-x mature leaf, color dark green dark greenx-18 mature leaf, color hue N/A N/A x-19 mature leaf, color intensitydark dark x-20 mature leaf, anthocyanin N/A N/A x-21 mature leaf,anthocyanin intensity N/A N/A 4-22 mature leaf, anthocyanin distributionN/A N/A 4-x mature leaf, anthocyanin concentration N/A N/A x-23 matureleaf, kind of anthocyanin N/A N/A 4-x mature leaf, leaf size large large4-24 mature leaf, glossiness, upper side moderate moderate 4-25 matureleaf, blistering very strong moderate x-26 mature leaf, blister sizelarge large 4-14 mature leaf, thickness thick thick 4-x mature leaf,trichomes absent absent x-15 mature leaf, attitude at harvest erecterect x-16 mature leaf, shape broad obtrullate broad obtrullate x-17mature leaf, shape at tip obtuse obtuse 5-x mature plant, spread 34.1 cm35.6 cm x-7 mature plant, diameter large large x-36 mature plant, heighttall tall x-37 mature plant, fasciation absent absent x-38 mature plant,fasciation intensity none none x-8 mature plant, head formation openhead open head x-9 mature plant, head type N/A N/A 5-x mature plant,head diameter 29.8 cm 27.8 cm 5-x mature plant, head shape elongateelongate x-13 mature plant, longitudinal cross-section narrow ellipticnarrow elliptic 5-11 mature plant, head size large large 5-x matureplant, heads/carton 24 heads 24 heads 5-x mature plant, head weight 779g 902 g 5-10 mature plant, head firmness medium medium 6-x mature plant,bottom shape rounded rounded 6-x mature plant, bottom midrib prominentlyraised moderately raised 7-x mature plant, core diameter 40 mm 43 m 7-xmature plant, head dia/core dia ratio 8.5 6.5 7-x mature plant, coreheight 78 mm 89 mm BOLTING 8-x bolting, first date Jul. 30, 2020 Jul.30, 2020 8-x bolting, days to bolting 65 days 55 days 8-x bolting classmedium medium x-35 bolting, stalk medium medium 8-x bolting, height 150cm 163 cm 8-x bolting, spread 40 cm 48 cm 8-x bolting, leaves curvedcurved 8-x bolting, margin entire entire 8-x bolting, color medium greenmedium green 8-x bolting terminal inflorescence present present 8-xbolting, lateral shoots present present 8-x bolting, basal side shootsabsent absent x-33, bolting, axillary sprouting medium medium x-34,bolting, harvest maturity medium medium *These are typical values.Values may vary due to environment. Other values that are substantiallyequivalent are also within the scope of the invention.

C. Breeding Romaine Lettuce Cv. Toro

One aspect of the current invention concerns methods for crossing theromaine lettuce cv. Toro with itself or a second plant and the seeds andplants produced by such methods. These methods can be used forpropagation of cv. Toro or can be used to produce hybrid lettuce seedsand the plants grown therefrom. Hybrid seeds are produced by crossingcv. Toro with second lettuce parent line.

The development of new varieties using one or more starting varieties iswell known in the art. In accordance with the invention, novel varietiesmay be created by crossing cv. Toro followed by multiple generations ofbreeding according to such well-known methods. New varieties may becreated by crossing with any second plant. In selecting such a secondplant to cross for the purpose of developing novel lines, it may bedesired to choose those plants which either themselves exhibit one ormore selected desirable characteristics or which exhibit the desiredcharacteristic(s) when in hybrid combination. Once initial crosses havebeen made, inbreeding and selection take place to produce new varieties.For development of a uniform line, often five or more generations ofselfing and selection are involved.

The breeding method employed was pedigree selection, using both singleplant selection and mass selection practices. The selection criteria forcv. Toro were to identify a romaine variety with acceptable size andweight, having resistance to lettuce necrotic stunt virus, as well asresistance to tipburn (enhanced translocation of calcium in youngdeveloping leaves).

In another embodiment, uniform lines of new varieties may also bedeveloped by way of double haploids. This technique allows the creationof true breeding lines without the need for multiple generations ofselfing and selection. In this manner true breeding lines can beproduced in as little as one generation. Haploid embryos may be producedfrom microspores, pollen, anther cultures, or ovary cultures. Thehaploid embryos may then be doubled autonomously, or by chemicaltreatments (e.g. colchicine treatment). Alternatively, haploid embryosmay be grown into haploid plants and treated to induce chromosomedoubling. In either case, fertile homozygous plants are obtained. Inaccordance with the invention, any of such techniques may be used inconnection with cv. Toro and progeny thereof to achieve a homozygousline.

New varieties may be created, for example, by crossing cv. Toro with anysecond plant and selection of progeny in various generations and/or bydoubled haploid technology. In choosing a second plant to cross for thepurpose of developing novel lines, it may be desired to choose thoseplants which either themselves exhibit one or more selected desirablecharacteristics or which exhibit the desired characteristic(s) inprogeny. After one or more lines are crossed, true-breeding lines may bedeveloped.

Backcrossing can also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait from one inbred or non-inbredsource to an inbred that lacks that trait. This can be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriate locusor loci for the trait in question. The progeny of this cross are thenmated back to the superior recurrent parent (A) followed by selection inthe resultant progeny for the desired trait to be transferred from thenon-recurrent parent. After five or more backcross generations withselection for the desired trait, the progeny are heterozygous for locicontrolling the characteristic being transferred, but are like thesuperior parent for most or almost all other loci. The last backcrossgeneration would be selfed to give pure breeding progeny for the traitbeing transferred.

The line of the present invention is particularly well suited for thedevelopment of new lines based on the elite nature of the geneticbackground of the line. In selecting a second plant to cross with cv.Toro for the purpose of developing novel lettuce lines, it willtypically be preferred to choose those plants which either themselvesexhibit one or more selected desirable characteristics or which exhibitthe desired characteristic(s) when in hybrid combination.

D. Performance Characteristics of Romaine Lettuce Cv. Toro

As described above, cv. Toro exhibits desirable performance traits. Theresults of an analysis of such traits are presented below.

TABLE 2 Evaluation of romaine cv. Ranger, cv. Lompoc, and cv. Toro andthe most similar cultivar, cv. River Road, for Head Diameter, HeadWeight, Head Height, Outer Leaf Blister, and Days Until Stem Reaches to15 cm, showing means, standard deviation, and range of variation at the95% confidence level. Trial Head Head Head Outer Leaf Days to NumberCultivar (cv.) Rep No. Diameter^(a) Weight^(b) Height^(c) Blister^(d) 15cm^(e) Trial 1: cv. Ranger Rep. 1 30.7 ± 0.4 957 ± 27.9 34.4 ± 0.7 2.2 ±0.1 64.7 ± 0.8 Evaluated Rep. 2 31.3 ± 0.4 1201 ± 28.1  34.9 ± 0.5 2.1 ±0.2 65.5 ± 0.7 Aug. 21, Average: 31.0 ± 0.4 1079 ± 28.0  34.7 ± 0.6 2.15± 0.2  65.1 ± 0.8 2019; cv. Lompoc Rep. 1 33.9 ± 0.5 935 ± 29.1 32.8 ±0.6 2.2 ± 0.2 66.1 ± 0.9 Chualar, CA Rep. 2 34.8 ± 0.4 988 ± 28.4 31.5 ±0.5 2.1 ± 0.2 63.9 ± 0.8 Average: 34.4 ± 0.5 962 ± 28.8 32.2 ± 0.6 2.15± 0.2  65.0 ± 0.9 cv. Toro Rep. 1 29.0 ± 0.5 744 ± 26.7 30.3 ± 0.5 2.2 ±0.2 67.3 ± 0.6 Rep. 2 29.6 ± 0.4 780 ± 28.2 29.6 ± 0.7 2.3 ± 0.1 65.8 ±0.8 Average: 29.3 ± 1.5 762 ± 27.5 30.0 ± 0.6 2.25 ± 0.2  66.6 ± 0.7 cv.River Road Rep. 1 27.7 ± 0.4 881 ± 27.2 36.2 ± 0.6 2.25 ± 0.2  66.6 ±0.7 Rep. 2 27.2 ± 0.4 928 ± 28.2 36.8 ± 0.6 0.3 ± 0.1 53.5 ± 0.5Average: 27.5 ± 0.4 905 ± 27.7 36.5 ± 0.6 0.35 ± 0.1  54.4 ± 0.6 Trial2: cv. Ranger Rep. 1 31.2 ± 0.5 1055 ± 27.5  34.5 ± 0.5 2.4 ± 0.2 64.6 ±0.7 Evaluated Rep. 2 31.4 ± 0.5 972 ± 27.7 34.7 ± 0.5 2.2 ± 0.1 65.1 ±0.7 Aug. 27, Average: 31.3 ± 0.5 979 ± 27.6 34.6 ± 0.5 2.3 ± 0.2 64.9 ±0.7 2020; cv. Lompoc Rep. 1 34.4 ± 0.5 971 ± 28.1 31.6 ± 0.7 2.2 ± 0.266.4 ± 0.8 Soledad, CA Rep. 2 34.1 ± 0.5 987 ± 28.2 32.2 ± 0.6 2.3 ± 0.265.9 ± 0.8 Average: 34.3 ± 0.5 979 ± 28.2 31.9 ± 0.7 2.25 ± 0.2  66.2 ±0.8 cv. Toro Rep. 1 30.3 ± 0.5 815 ± 28.6 29.9 ± 0.7 2.1 ± 0.1 64.3 ±0.6 Rep. 2 30.0 ± 0.4 777 ± 28.4 28.8 ± 0.7 2.3 ± 0.2 65.8 ± 0.8Average: 30.2 ± 0.5 796 ± 28.5 29.4 ± 0.7 2.2 ± 0.2 65.1 ± 0.7 cv. RiverRoad Rep. 1 27.6 ± 0.4 878 ± 27.8 35.7 ± 0.7 0.4 ± 0.1 54.2 ± 0.7 Rep. 228.2 ± 0.5 899 ± 28.4 36.5 ± 0.6 0.3 ± 0.1 55.7 ± 0.6 Average: 27.9 ±0.5 889 ± 28.1 36.5 ± 0.6 0.35 ± 0.1  55.0 ± 0.7 Range of variationamong means of statistically significant differences at the 95% levelusing

 confidence interval [CI = mean ± (SDXSE)]: cv. Ranger 30.6 to 31.81039.8 to 1052.2 34.5 to 34.7 2.16 to 2.26 64.8 to 65.9 cv. Lompoc 33.7to 34.9 963.6 to 976.4 31.9 to 32.1 2.15 to 2.25 65.3 to 66.2 cv. Toro29.1 to 30.3 772.7 to 785.3 29.6 to 29.8 2.18 to 2.28 65.7 to 66.5 cv.River Road 27.1 to 28.3 890.8 to 903.2 36.2 to 36.5 0.33 to 0.37 54.3 to54.8 ^(a)Mean head diameter using two sowing dates of 20 plants perreplication in cm ± standard deviation. ^(b)Mean head weight using twosowing dates of 20 plants per replication in grams ± standard deviation.^(c)Mean number of days until stem reaches 15 cm using two replicationsof 20 plants each ± standard deviation. ^(d)Mean of the number of leafblisters per square centimeter, using two sowing dates of 20 plants perreplication ± standard deviation. ^(e)Mean number of days until stemreaches 15 cm using two replications of 20 plants each, ±standarddeviation.

indicates data missing or illegible when filed

In replicated field trials of cv. Toro, during 2019 and 2020, coveringgenerations F8 and F9, no genetic variants nor off-types have beenobserved in more than 5,000 individuals, indicating these lines aregenetically uniform and stable.

In replicated field trials, heads of cv. Toro were consistently smallerin diameter than cvs. Lompoc and Ranger, (29.7 cm, 34.4 cm, 31.2 cm,respectively), but larger than cv. River Road (27.7 cm; Table 2). Cv.Toro was lighter in weight than cvs. Ranger, Lompoc, and River Road (779g vs. 1,047 g, 971 g, 897 g, respectively). When measured for headheight, cv. River Road was tallest, followed by cvs. Ranger and Lompoc,(34.7 cm, 33.4 cm, 36.3 cm, respectively), with cv. Toro being theshortest (29.7 cm). Measurements of outer leaf blistering indicated cvs.Toro, Ranger, and Lompoc had more blisters per square centimeter thancv. River Road (2.25, 2.2, 2.3, and 0.35, respectively), whilemeasurements of days for the stem to reach 15 cm in height did notdiffer greatly different between cvs. Toro, Ranger, and Lompoc, (65.8,65.4, 65.6, respectively), however were later when compared to cv. RiverRoad (54.7), indicating cvs. Toro, Ranger, and Lompoc take longer toinitiate stem elongation (bolting).

The data presented here, for the four traits mentioned above, arestatistically different at the 95% confidence level, exhibiting a rangeof means for head diameter, head weight, head height, outer leafblistering, and days to 15 cm for the four cultivars evaluated (Table2). Therefore, these data illustrate that cv. Toro differedsignificantly from cvs. Ranger and Lompoc, while also differing from themost similar variety, cv. River Road, in field trials conducted in 2019and 2020. For these reasons, romaine cv. Toro illustrates uniquecharacteristics not observed during evaluations of the most similarvariety, cv. River Road, nor with cvs. Ranger and Lompoc.

E. Further Embodiments of the Invention

In specific embodiments, the invention provides plants modified toinclude at least a first desired heritable trait. Such plants may, inone embodiment, be developed by a plant breeding technique calledbackcrossing, wherein essentially all of the desired morphological andphysiological characteristics of a variety are recovered in addition toa genetic locus transferred into the plant via the backcrossingtechnique. The terms converted plant or single locus converted plant asused herein refers to those lettuce plants which are developed by aplant breeding technique called backcrossing, wherein essentially all ofthe desired morphological and physiological characteristics of a varietyare recovered in addition to the single locus transferred into thevariety via the backcrossing technique. By essentially all of themorphological and physiological characteristics, it is meant that thecharacteristics of a plant are recovered that are otherwise present whencompared in the same environment, other than an occasional variant traitthat might arise during backcrossing or direct introduction of atransgene. It is understood that a locus introduced by backcrossing mayor may not be transgenic in origin, and thus the term backcrossingspecifically includes backcrossing to introduce loci that were createdby genetic transformation.

Backcrossing methods can be used with the present invention to improveor introduce a characteristic into the present variety. The parentallettuce plant which contributes the locus for the desired characteristicis termed the nonrecurrent or donor parent. This terminology refers tothe fact that the nonrecurrent parent is used one time in the backcrossprotocol and therefore does not recur. The parental lettuce plant towhich the locus or loci from the nonrecurrent parent are transferred isknown as the recurrent parent as it is used for several rounds in thebackcrossing protocol.

In a typical backcross protocol, the original variety of interest(recurrent parent) is crossed to a second variety (nonrecurrent parent)that carries the single locus of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a lettuce plant isobtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, in addition to the single transferred locus fromthe nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single locus of the recurrent variety ismodified or substituted with the desired locus from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered to determine an appropriate testing protocol.Although backcrossing methods are simplified when the characteristicbeing transferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

In one embodiment, progeny lettuce plants of a backcross in which cv.Toro is the recurrent parent comprise (i) the desired trait from thenon-recurrent parent and (ii) all of the physiological and morphologicalcharacteristics of romaine lettuce cv. Toro as determined at the 5%significance level when grown in the same environmental conditions.

Lettuce varieties can also be developed from more than two parents. Thetechnique, known as modified backcrossing, uses different recurrentparents during the backcrossing. Modified backcrossing may be used toreplace the original recurrent parent with a variety having certain moredesirable characteristics or multiple parents may be used to obtaindifferent desirable characteristics from each.

With the development of molecular markers associated with particulartraits, it is possible to add additional traits into an established germline, such as represented here, with the end result being substantiallythe same base germplasm with the addition of a new trait or traits.Molecular breeding, as described in Moose and Mumm, 2008 (PlantPhysiology, 147: 969-977), for example, and elsewhere, provides amechanism for integrating single or multiple traits or QTL into an eliteline. This molecular breeding-facilitated movement of a trait or traitsinto an elite line may encompass incorporation of a particular genomicfragment associated with a particular trait of interest into the eliteline by the mechanism of identification of the integrated genomicfragment with the use of flanking or associated marker assays. In theembodiment represented here, one, two, three or four genomic loci, forexample, may be integrated into an elite line via this methodology. Whenthis elite line containing the additional loci is further crossed withanother parental elite line to produce hybrid offspring, it is possibleto then incorporate at least eight separate additional loci into thehybrid. These additional loci may confer, for example, such traits as adisease resistance or a fruit quality trait. In one embodiment, eachlocus may confer a separate trait. In another embodiment, loci may needto be homozygous and exist in each parent line to confer a trait in thehybrid. In yet another embodiment, multiple loci may be combined toconfer a single robust phenotype of a desired trait.

Many single locus traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single locus traits may or may not betransgenic; examples of these traits include, but are not limited to,male sterility, herbicide resistance, resistance to bacterial, fungal,or viral disease, insect resistance, restoration of male fertility,modified fatty acid or carbohydrate metabolism, and enhanced nutritionalquality. These comprise genes generally inherited through the nucleus.

Direct selection may be applied where the single locus acts as adominant trait. An example of a dominant trait is the downy mildewresistance trait. For this selection process, the progeny of the initialcross are sprayed with downy mildew spores prior to the backcrossing.The spraying eliminates any plants which do not have the desired downymildew resistance characteristic, and only those plants which have thedowny mildew resistance gene are used in the subsequent backcross. Thisprocess is then repeated for all additional backcross generations.

Selection of lettuce plants for breeding is not necessarily dependent onthe phenotype of a plant and instead can be based on geneticinvestigations. For example, one can utilize a suitable genetic markerwhich is closely genetically linked to a trait of interest. One of thesemarkers can be used to identify the presence or absence of a trait inthe offspring of a particular cross, and can be used in selection ofprogeny for continued breeding. This technique is commonly referred toas marker assisted selection. Any other type of genetic marker or otherassay which is able to identify the relative presence or absence of atrait of interest in a plant can also be useful for breeding purposes.Procedures for marker assisted selection applicable to the breeding oflettuce are well known in the art. Such methods will be of particularutility in the case of recessive traits and variable phenotypes, orwhere conventional assays may be more expensive, time consuming orotherwise disadvantageous. Types of genetic markers which could be usedin accordance with the invention include, but are not necessarilylimited to, Simple Sequence Length Polymorphisms (SSLPs) (Williams etal., Nucleic Acids Res., 1 8:6531-6535, 1990), Randomly AmplifiedPolymorphic DNAs (RAPDs), DNA Amplification Fingerprinting (DAF),Sequence Characterized Amplified Regions (SCARs), Arbitrary PrimedPolymerase Chain Reaction (AP-PCR), Amplified Fragment LengthPolymorphisms (AFLPs) (EP 534 858, specifically incorporated herein byreference in its entirety), and Single Nucleotide Polymorphisms (SNPs)(Wang et al., Science, 280:1077-1082, 1998).

F. Plants Derived from Romaine Lettuce Cv. Toro by Genetic Engineering

Many useful traits that can be introduced by backcrossing, as well asdirectly into a plant, are those which are introduced by genetictransformation techniques. Genetic transformation may therefore be usedto insert a selected transgene into the lettuce line of the invention ormay, alternatively, be used for the preparation of transgenes which canbe introduced by backcrossing. Methods for the transformation of plants,including lettuce, are well known to those of skill in the art.

Vectors used for the transformation of lettuce cells are not limited solong as the vector can express an inserted DNA in the cells. Forexample, vectors comprising promoters for constitutive gene expressionin lettuce cells (e.g., cauliflower mosaic virus 35S promoter) andpromoters inducible by exogenous stimuli can be used. Examples ofsuitable vectors include pBI binary vector. The “lettuce cell” intowhich the vector is to be introduced includes various forms of lettucecells, such as cultured cell suspensions, protoplasts, leaf sections,and callus.

A vector can be introduced into lettuce cells by known methods, such asthe polyethylene glycol method, polycation method, electroporation,Agrobacterium-mediated transfer, particle bombardment and direct DNAuptake by protoplasts. See, e.g., Pang et al. (The Plant J., 9, 899-909,1996).

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wound tissues ina controlled manner. An example of electroporation of lettuceprotoplasts is presented in Chupeau et al. (Bio/Tech., 7:503-508, 1989).

A particularly efficient method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesare coated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. For the bombardment, cells in suspensionare concentrated on filters or solid culture medium. Alternatively,immature embryos or other target cells may be arranged on solid culturemedium. The cells to be bombarded are positioned at an appropriatedistance below the macroprojectile stopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System, whichcan be used to propel particles coated with DNA or cells through ascreen, such as a stainless steel or Nytex screen, onto a surfacecovered with target lettuce cells. The screen disperses the particles sothat they are not delivered to the recipient cells in large aggregates.It is believed that a screen intervening between the projectileapparatus and the cells to be bombarded reduces the size of projectilesaggregate and may contribute to a higher frequency of transformation byreducing the damage inflicted on the recipient cells by projectiles thatare too large.

Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species. Examples involvingmicroprojectile bombardment transformation with lettuce can be found in,for example, Elliott et al. (Plant Cell Rep., 18:707-714, 2004) andMolinier et al. (Plant Cell Rep., 21:251-256, 2002).

Agrobacterium-mediated transfer is another widely applicable system forintroducing gene loci into plant cells. An advantage of the technique isthat DNA can be introduced into whole plant tissues, thereby bypassingthe need for regeneration of an intact plant from a protoplast. ModernAgrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations(Klee et al., Bio-Technology, 3(7):637-642, 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described haveconvenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes. Additionally, Agrobacterium containing both armed anddisarmed Ti genes can be used for transformation.

In those plant strains where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene locus transfer. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (Fraley et al., Bio/Technology, 3:629-635, 1985; U.S.Pat. No. 5,563,055). For example, U.S. Pat. No. 5,349,124 describes amethod of transforming lettuce plant cells using Agrobacterium-mediatedtransformation. By inserting a chimeric gene having a DNA codingsequence encoding for the full-length B.t. toxin protein that expressesa protein toxic toward Lepidopteran larvae, this methodology resulted inlettuce having resistance to such insects.

Transformation of plant protoplasts also can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., Mol. Gen. Genet., 199:183-188, 1985; Omirulleh et al.,Plant Mol. Biol., 21(3):415-428, 1993; Fromm et al., Nature,312:791-793, 1986; Uchimiya et al., Mol. Gen. Genet., 204:204, 1986;Marcotte et al., Nature, 335:454, 1988). Transformation of plants andexpression of foreign genetic elements is exemplified in Choi et al.(Plant Cell Rep., 13: 344-348, 1994) and Ellul et al. (Theon. Appl.Genet., 107:462-469, 2003).

A number of promoters have utility for plant gene expression for anygene of interest including but not limited to selectable markers,scoreable markers, genes for pest tolerance, disease resistance,nutritional enhancements and any other gene of agronomic interest.Examples of constitutive promoters useful for lettuce plant geneexpression include, but are not limited to, the cauliflower mosaic virus(CaMV) P-35S promoter, which confers constitutive, high-level expressionin most plant tissues (see, e.g., Odel et al., Nature, 313:810, 1985),including monocots (see, e.g., Dekeyser et al., Plant Cell, 2:591, 1990;Terada and Shimamoto, Mol. Gen. Genet., 220:389, 1990); a tandemlyduplicated version of the CaMV 35S promoter, the enhanced 35S promoter(P-e35S) the nopaline synthase promoter (An et al., Plant Physiol.,88:547, 1988), the octopine synthase promoter (Fromm et al., Plant Cell,1:977, 1989); and the figwort mosaic virus (P-FMV) promoter as describedin U.S. Pat. No. 5,378,619 and an enhanced version of the FMV promoter(P-eFMV) where the promoter sequence of P-FMV is duplicated in tandem,the cauliflower mosaic virus 19S promoter, a sugarcane bacilliform viruspromoter, a commelina yellow mottle virus promoter, and other plant DNAvirus promoters known to express in plant cells.

A variety of plant gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals can beused for expression of an operably linked gene in plant cells, includingpromoters regulated by (1) heat (Callis et al., Plant Physiol., 88:965,1988), (2) light (e.g., pea rbcS-3A promoter, Kuhlemeier et al., PlantCell, 1:471, 1989; maize rbcS promoter, Schaffner and Sheen, Plant Cell,3:997, 1991; or chlorophyll a/b-binding protein promoter, Simpson etal., EMBO 1, 4:2723, 1985), (3) hormones, such as abscisic acid(Marcotte et al., Plant Cell, 1:969, 1989), (4) wounding (e.g., wunl,Siebertz et al., Plant Cell, 1:961, 1989); or (5) chemicals such asmethyl jasmonate, salicylic acid, or Safener. It may also beadvantageous to employ organ-specific promoters (e.g., Roshal et al.,EMBO 1, 6:1155, 1987; Schernthaner et al., EMBO J., 7:1249, 1988; Bustoset al., Plant Cell, 1:839, 1989).

Exemplary nucleic acids which may be introduced to the lettuce lines ofthis invention include, for example, DNA sequences or genes from anotherspecies, or even genes or sequences which originate with or are presentin the same species, but are incorporated into recipient cells bygenetic engineering methods rather than classical reproduction orbreeding techniques. However, the term “exogenous” is also intended torefer to genes that are not normally present in the cell beingtransformed, or perhaps simply not present in the form, structure, etc.,as found in the transforming DNA segment or gene, or genes which arenormally present and that one desires to express in a manner thatdiffers from the natural expression pattern, e.g., to over-express.Thus, the term “exogenous” gene or DNA is intended to refer to any geneor DNA segment that is introduced into a recipient cell, regardless ofwhether a similar gene may already be present in such a cell. The typeof DNA included in the exogenous DNA can include DNA which is alreadypresent in the plant cell, DNA from another plant, DNA from a differentorganism, or a DNA generated externally, such as a DNA sequencecontaining an antisense message of a gene, or a DNA sequence encoding asynthetic or modified version of a gene.

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a lettuce plant according to theinvention. Non-limiting examples of particular genes and correspondingphenotypes one may choose to introduce into a lettuce plant include oneor more genes for insect tolerance, such as a Bacillus thuringiensis(B.t.) gene, pest tolerance such as genes for fungal disease control,herbicide tolerance such as genes conferring glyphosate tolerance, andgenes for quality improvements such as yield, nutritional enhancements,environmental or stress tolerances, or any desirable changes in plantphysiology, growth, development, morphology or plant product(s). Forexample, structural genes would include any gene that confers insecttolerance including but not limited to a Bacillus insect control proteingene as described in WO 99/31248, herein incorporated by reference inits entirety, U.S. Pat. No. 5,689,052, herein incorporated by referencein its entirety, U.S. Pat. Nos. 5,500,365 and 5,880,275, hereinincorporated by reference it their entirety. In another embodiment, thestructural gene can confer tolerance to the herbicide glyphosate asconferred by genes including, but not limited to Agrobacterium strainCP4 glyphosate resistant EPSPS gene (aroA:CP4) as described in U.S. Pat.No. 5,633,435, herein incorporated by reference in its entirety, orglyphosate oxidoreductase gene (GOX) as described in U.S. Pat. No.5,463,175, herein incorporated by reference in its entirety.

Alternatively, the DNA coding sequences can affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see, for example, Birdet al., Biotech. Gen. Engin. Rev., 9:207, 1991). The RNA could also be acatalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desiredendogenous mRNA product (see for example, Gibson and Shillito, Mol.Biotech., 7:125,1997). Thus, any gene which produces a protein or mRNAwhich expresses a phenotype or morphology change of interest is usefulfor the practice of the present invention.

G. Definitions

In the description and tables herein, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided:

Allele: Any of one or more alternative forms of a gene locus, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny, for example a first-generation hybrid (F₁), back to one of theparents of the hybrid progeny. Backcrossing can be used to introduce oneor more single locus conversions from one genetic background intoanother.

Converted (Conversion) Plant: Plants which are developed by a plantbreeding technique called backcrossing, wherein essentially all of thedesired morphological and physiological characteristics of a lettuceline are recovered in addition to the trait transferred into the varietyvia the backcrossing technique and/or by genetic transformation.

Crossing: The mating of two parent plants.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Diploid: A cell or organism having two sets of chromosomes.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a cytoplasmic or nuclear genetic factor conferring malesterility or a chemical agent.

Enzymes: Molecules which can act as catalysts in biological reactions.

F₁ Hybrid: The first-generation progeny of the cross of two nonisogenicplants.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Quantitative Trait Loci (QTL): Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Regeneration: The development of a plant from tissue culture.

Royal Horticultural Society (RHS) color chart value: The RHS color chartis a standardized reference which allows accurate identification of anycolor. A color's designation on the chart describes its hue, brightnessand saturation. A color is precisely named by the RHS color chart byidentifying the group name, sheet number and letter, e.g., Yellow-OrangeGroup 19A or Red Group 41B.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Substantially Equivalent: A characteristic that, when compared, does notshow a statistically significant difference (e.g., p=0.05) from themean.

Tetraploid: A cell or organism having four sets of chromosomes.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic locus comprising a sequence which has beenintroduced into the genome of a lettuce plant by transformation.

Triploid: A cell or organism having three sets of chromosomes.

H. Deposit Information

A deposit of romaine lettuce cv. Toro, disclosed above and recited inthe claims, has been made with the American Type Culture Collection(ATCC), 10801 University Blvd., Manassas, Va. 20110-2209. The date ofdeposit was ______. Upon issuance of a patent, all restrictions upon thedeposit will be removed, and the deposit is intended to meet all of therequirements of 37 C.F.R. § 1.801-1.809. The accession number for thosedeposited seeds of romaine lettuce cv. Toro is ATCC Accession No.PTA-______. The deposit will be maintained in the depository for aperiod of 30 years, or 5 years after the last request, or for theeffective life of the patent, whichever is longer, and will be replaced,if necessary, during that period.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

All references cited herein are hereby expressly incorporated herein byreference.

What is claimed is:
 1. A seed of romaine lettuce cv. Toro, a sample of seed of said line having been deposited under ATCC Accession Number PTA-______.
 2. A plant grown from the seed of claim
 1. 3. A plant part of the plant of claim
 2. 4. The plant part of claim 3, wherein said part is selected from the group consisting of a pollen, an ovule and a cell.
 5. A lettuce plant, or a part thereof, having all the physiological and morphological characteristics of the lettuce plant of claim
 2. 6. A tissue culture of regenerable cells of romaine lettuce cv. Toro, a sample of seed of said line having been deposited under ATCC Accession Number PTA-______.
 7. The tissue culture according to claim 6, comprising cells or protoplasts from a plant part selected from the group consisting of embryos, meristems, cotyledons, pollen, leaves, anthers, roots, root tips, pistil, flower, seed and stalks.
 8. A lettuce plant regenerated from the tissue culture of claim 6, wherein the regenerated plant expresses all of the physiological and morphological characteristics of romaine lettuce cv. Toro, a sample of seed of said line having been deposited under ATCC Accession Number PTA-______.
 9. A method of producing lettuce seed, comprising crossing the plant of claim 2 with a second lettuce plant.
 10. The method of claim 9, wherein the plant of romaine lettuce cv. Toro is the female parent.
 11. The method of claim 9, wherein the plant of romaine lettuce cv. Toro is the male parent.
 12. An F₁ hybrid seed produced by the method of claim
 9. 13. An F₁ hybrid plant produced by growing the seed of claim
 12. 13. A method for producing a seed of a romaine lettuce cv. Toro-derived lettuce plant comprising the steps of: (a) crossing a lettuce plant of romaine lettuce cv. Toro, a sample of seed of said line having been deposited under ATCC Accession Number PTA-______, with a second lettuce plant; and (b) allowing seed of a cv. Toro-derived lettuce plant to form.
 15. The method of claim 14, further comprising the steps of: (c) crossing a plant grown from said cv. Toro-derived lettuce seed with itself or a second lettuce plant to yield additional cv. Toro-derived lettuce seed; (d) growing said additional cv. Toro-derived lettuce seed of step (c) to yield additional cv. Toro-derived lettuce plants; and (e) repeating the crossing and growing steps of (c) and (d) to generate further cv. Toro-derived lettuce plants.
 16. A method of vegetatively propagating a plant of lettuce cv. Toro comprising the steps of: (a) collecting tissue capable of being propagated from a plant of lettuce cv. Toro, a sample of seed of said line having been deposited under ATCC Accession Number PTA-______; and (b) producing at least a first rooted plant from said tissue.
 17. A process of producing a conversion of romaine lettuce cv. Toro comprising at least one new trait, the process comprising: (a) crossing a plant of romaine lettuce cv. Toro, wherein a sample of seed of said line has been deposited under ATCC Accession Number PTA-______, with a lettuce plant that comprises at least one new trait to produce progeny seed; (b) harvesting and planting the progeny seed to produce at least one progeny plant of a subsequent generation, wherein the progeny plant comprises the at least one new trait; (c) crossing the progeny plant with a plant of romaine lettuce cv. Toro to produce backcross progeny seed; (d) harvesting and planting the backcross progeny seed to produce at least one backcross progeny plant; and (e) repeating steps (c) and (d) for at least three additional generations to produce a converted plant of romaine lettuce cv. Toro, wherein the converted plant of romaine lettuce cv. Toro comprises the at least one new trait.
 18. A converted lettuce plant produced by the method of claim
 17. 19. A method of producing a plant of romaine lettuce cv. Toro comprising an added desired trait, the method comprising introducing a transgene conferring the desired trait into a plant of romaine lettuce cv. Toro, wherein a sample of seed of said line has been deposited under ATCC Accession Number PTA-______.
 20. A plant produced by the method of claim
 19. 21. A method of producing food comprising: (a) obtaining the plant of claim 2, and (b) collecting leaf tissue from the plant, wherein the leaf tissue is capable of use as food. 